Apoptosis regulating composition

ABSTRACT

An object of the invention is to provide an apoptosis regulating composition. According to the invention, an apoptosis regulating composition is provided which comprises, as an active ingredient, at least one carbostyril derivatives of general formula (1) ##STR1## and salts thereof.

This is a divisional of application Ser. No. 07/989,028, now abandoned,filed as PCT/JP92/00841, Jul. 2, 1992.

The present invention relates to a novel apoptosis regulatingcomposition.

The apoptosis regulating composition of the invention comprises, as anactive ingredient, at least one member of carbostyril derivativerepresented by the following general formula (1) (hereinafter referredto as compound (1)) and salts thereof. ##STR2## wherein R is a benzoylgroup which may optionally have lower alkoxy groups on the phenyl ringas substituents and the carbon-carbon bond in the 3 and 4 positions ofthe carbostyril skeleton is a single bond or a double bond.

Regarding the above compound (1) and the processes available forproduction thereof, the descriptions in the Japanese Examined PatentPublication No. 43747/1989 (corresponding to U.S. Pat. No. 4,415,572)can be consulted. It is also known that the compound (1) is useful ascardiotonic.

The inventor of this invention did further research into the compound(1) and found that they have apoptosis regulating or modifying(suppressing or promoting) activity, including anticancer activity andcell differentiation inducing activity, among others, which can hardlybe anticipated from their known activities mentioned above.

Meanwhile, it is said that two types of mechanism are involved in celldeath. One is a classical type of cell death called necrosis.Morphologically, necrosis is characterized by marked swelling ofmitochondria, swelling of cytoplasm and nuclear alteration, followed bycell destruction and autolysis. It occurs passively or incidentally.Tissue necrosis is generally caused by physical trauma to cells or achemical poison, for instance.

Another type of cell death is called apoptosis (programmed cell death)[Kerr, J. F. R. and Wyllie, A. H., Br. J. Cancer, 265, 239 (1972)]. Itis said that apoptosis can occur under various physiological conditions.Morphologically, apoptosis is characterized by loss of contact withneighboring cells, concentration of cytoplasm, endonucleaseactivity-associated chromatin condensation and pyknosis, andsegmentation of the nucleus, among others. Disappearance of microvillifrom the cell surface and smoothening of the cell surface (vesicleformation on the cell surface: membrane blebbing) are also observed.Fragmentation of the nucleosome unit of DNA into DNA fragments 180-200bases in size due to endonuclease activation is further observable.Final fragments of apoptotic body cells are phagocytosed by neighboringcells. This is the mechanism discussed by Duvall and Wyllie [Duvall, E.and Wyllie, A. H., Immunology Today, 7 (4), 115-119 (1986); Science,245, 301-305 (1989)]. Wyllie further reported thatglucocorticoid-induced apoptosis of thymocytes involves intracellularendonuclease activation [Wyllie, A. H., Nature, 284, 555-556 (1986)].Endonuclease activity causes fragmentation, to the oligonucleotidelevel, of DNA in cells undergoing apoptosis and this can be readilyconfirmed by agarose gel electrophoresis.

Apoptosis can be considered as preprogrammed cell death seen in theprocess of development, differentiation, or turnover of tissues [Wyllie,A. H., et al., Int. Rev. Cytol., 68, 251-306 (1980)].

In thymocytes, an increase in calcium level in the calcium ionophore oran increase in cAMP level leads to promotion of that DNA fragmentationwhich is characteristic of the above-mentioned apoptosis [Wyllie, A. H.et al., J. Pathol., 142, 67-77 (1984)] and, therefore, it is supposedthat the calcium ion and/or cAMP be involved in the mechanisms ofapoptosis. As an example so far reported, there may be mentionedapoptosis of HL-60 cells whose differentiation is induced by retinoicacid or the calcium ionophore [Martin, S. J., et al., J. Immunol., 145,1859-1867 (1990); Martin, S. J. et al., Clin. Exp. Immunol., 79, 448-453(1990)].

Reportedly, apoptosis occurs not only upon physiological cell death inthe process of embryogenesis and physiological death of normal cells inactive cell cycle (e.g. liver, adrenal cortex and prostate cells) butalso is induced by glucocorticoid treatment, cell injury by cytotoxic Tcells, atrophy of hormone-dependent tissues, irradiation, NK cells,killer cells, tumor necrosis factor (TNF), lymphotoxin (LT), othercytokines, etc. [Wyllie, A. H. et al., Int. Rev. Cytol., 68, 251 (1980);Duvall, E. and Wyllie, A. H., Immunology Today, 7, 115-119 (1986);Sellins, K. S., et al., J. Immunol., 139, 3199 (1987); Yamada, T., etal., Int. J. Radiat. Biol., 53, 65 (1988); Wyllie, A. H., Nature, 284,555 (1980); Schmid, D. S., et al., Proc. Natl. Acad. Sci. U.S.A., 83,1881-1885 (1986); John, C., et al., J. Immunol., 129 (4), 1782-1787(1982); Howell, D. M., et al., J. Immunol., 140, 689-692 (1988);Gillian, B., et al., Eur. J. Immunol., 17, 689-693 (1987)]. In addition,apoptosis is also inducible by some antibodies, for example anti-CD3,anti-APO-I, and anti-Fas antibodies [Trauth, B. C., et al., Science,245, 301-305 (1989); Smith, C. A., et al. Nature, 337, 181-184 (1989);Tadakuma, T., et al., Eur. J. Immunol., 20, 779 (1990)] and, further,apoptosis has been confirmed in the findings of Nakamura et al. asobtained in spontaneous regression of malignant tumor [Nakamura, Y., etal., Rinsho Hifuka (Jpn, J. Clin, Dermatol.), 35 (4), 289-295 (1981)].

On the other hand, actinomycin D (an RNA synthesis inhibitor),cycloheximide (a protein synthesis inhibitor) and calcium ion (Ca²⁺)chelating agents, among others, have been reported as being capable ofrepressing apoptosis and, in addition, cyclosporin A (an immunosuppressant), hematopoietic system cytokines [IL-3, GM-CSF (granulocytemacrophage colony stimulating factor), G-CSF (granulocyte colonystimulating factor)], IL-2, bcl-2 gene product, and the like canreportedly repress apoptosis [Cohen, J. J., J. Immunol., 132, 38 (1984);Wyllie, A. H., et al., J. Pathol., 142, 67 (1984); Shi, Y., et al.,Nature, 339, 625 (1989); Williams, G. L., et al., Nature, 343, 76(1990); Nielo, M. A., J. Immunol., 143, 4166 (1989); Vaux, D. L., etal., Nature, 335, 1440 (1988)]. While, for cycloheximide and actinomycinD, there is a report describing apoptosis induction in acute leukemiacells by cycloheximide, in small intestine crypt cells by actinomycin D,and in HL-60 cells by both [Martin, S. J., et al., J. Immunol., 145,1859-1867 (1990)]. On the other hand, it is reported that cycloheximiderather suppresses, and actinomycin D potentiates, apoptosis of thelymphocytic tumor cells which are present before X-ray radiation and areincreased by X-ray radiation. Therefore, it is suggested that the kindof cells, conditions, and other mechanisms be involved in thesuppression or promotion of apoptosis [Igarashi, T., et al., NipponKetsueki Gakkaishi (Acta Hematol. Jpn.), 51 (2), 144 (1988)]. At anyrate, it is currently considered that the differentiation, growth andmaturation of cells are closely associated with apoptosis and thatsubstances capable of playing some or other part in such celldifferentiation, growth or the like are associated with apoptosis aswell.

Recently, cancer treatment with anti-Apo-I antibody has been attemptedas an apoptosis-related therapy. Among the myelodysplastic syndrome(MDS), refractory anemia (RA) and refractory anemia with ringsideroblast (RARS) in which pancytopenia is predominant shouldpreferably be treated with a combination of retinoic acid or vitamin D₃,which is a differentiation inducer for hemopoietic cells, and GM-CSF orIL-3 as an apoptosis regulating agent which suppresses excessiveapoptosis of platelet producing cells whereas, in RAEB (refractoryanemia with excess of blasts) and RAEB-t (RAEB in transformation) inwhich blast cell growth is active, retinoic acid and vitamin D₃ are saidto act as differentiation inducing agents, which induce differentiationof blast cells into mature blood cells, and etoposide and aclarubicinare said to act as apoptosis regulating agents, which suppress blastcell growth (thereby promote apoptosis) [Shibuya, T., J. Clin. Exp,Med., 160 (5), 319-323 (1992)].

Murakami et al. reported that about half of transgenic mice expressinganti-erythrocyte autoantibody manifest autoimmune diseases as a resultof loss of self tolerance and that this is due to deficiency in abilityto eliminate autoantibodies producing cells as resulting from apoptosisinduction by self antigen-autoantibody producing cells reactions as innormal mice [Murakami, M., et al., Nature, 357, 77-80 (1992)].

Watanabe-Fukunaga et al. suggest that, for MRL lpr/lpr mice, Fasmoleculs relating to apoptosis has abnormality and the negative relation(apoptosis) mechanism of autoreactive T-cells does not work properly inthymus. Consequently, autoimmune disease occur [Watanabe-Fukunaga, R.,et al., Nature, 356, 314-317 (1992)].

According to Montagnier et al., apoptotic DNA bands are observed in Tlymphocyte extracts from HIV-infected patients. This phenomenon isobserved in 90% of asymptomatic HIV-infected patients and in 100% ofAIDS patients and of ARC (AIDS-related complex) patients, indicatingincreased apoptosis induction in HIV-infected patients as well[Montagnier, L., et al., Sixieme Colloque des Cent Gardes, 9-17 (1991)].

As regards development stage cell death in chickens, administration inadvance of NGF (nerve growth factor; a protein that promotes cellhypertrophy and nerve fiber elongation in the nerve cell ganglion) canresult in complete inhibition of nerve cell death in that developmentstage [Hamburger, V., et al., J. Neurosci., 1, 60 (1981)] whileadministration of an antibody to NGF conversely leads to loss of about90% of juvenile sympathetic nerve cells [Levi-Montalchini, R. andBooker, B., Proc. Natl. Acad. Sci. U.S.A., 46, 384 (1960)].

Clark classified spontaneous neuronal deaths into three types andidentified type I as apoptosis since, in type I neuronal death,morphological characteristics are identical with those in apoptosis andsince type I cell death, together with DNA fragmentation, is involved inthe cell death caused by deprivation of the growth factor [Clark, P. G.H., Anat. Embryol., 181, 195 (1990); J. Neurosci., 1, 60 (1981); Proc.Natl. Acad. Sci. U.S.A., 46, 384 (1960); Rawson, C. L., et al., J. Cell.Biol., 113, 671 (1991)].

According to a report by Edwards et al., NGF can inhibit programmeddeath of sympathetic nerve cells, hence NGF can presumably controlapoptosis [Edwards, S. N., et al., J. Neurochemistry, 57 (6), 2140-2143(1991)].

According to Fischer et al., aged rats with learning disorder, whenadministered with NGF, can recover from learning disorder as a result ofsaid NGF acting on forebrain basal field cholinergic nerve cells whichare known to be found damaged in Alzheimer's disease [Fischer, W., etal., Nature, 329, 65 (1987); Bardel Y.-A., Neuron, 2, 1525 (1989);Hatanaka, H., Develop. Brain Res., 30, 47 (1986); Hatanaka, H., et al.,Develop. Brain Res., 39, 85 (1988)]. Hatanaka et al. suggest thepossibility that NGF can be effective in differentiation, maturation,life supporting and prevention of aging, protect nerve cells fromdamaging, promote recovery of damaged nerve cells and inhibit nerve celldeath in nervous diseases associated with aging of the brain, inparticular in Alzheimer's disease [Hatanaka, H., Taisha (Metabolism),28, 891-899 (1991)].

For hepatic lesion of drug resistant virus hepatitis, acceleration ofapoptosis which is direct or through the immune system, is considered tobe involved in hepatic lesion.

On the other hand, it is known that, in the liver, mitogens induce thegrowth of hepatocytes to produce a hyperplastic state, and this state isnormalized by falling off and necrosis, i.e. apoptosis, of hepatocytes[Kerr, J. F., et al., Br. J. Cancer, 26, 239-257 (1972)]. As far as theliver is concerned, apoptosis is observable in hepatic hyperplasia,hyperplastic tuberculation and hepatic cancer, among others [Columbano,A., et al., Lab. Invest., 52, 670-675 (1985); Columbano, A., et al., Am.J. Pathol., 116, 441-446 (1984)] while, according to Kerr et al.,apoptosis is not accompanied by inflammation or fibroplasia [Kerr, J.F., et al., Lancet, 2, 827-828 (1979)].

In view of the reports cited above, the present inventors consider thatpatients with hepatitis, whether acute or chronic, may be cured whenapoptosis is inhibited. They further consider that, in patients in theprocess of transition from chronic hepatitis to hepatic cirrhosis andfurther to hepatic cancer, apoptosis is in a controlled state and thuscytotoxic T cells can induce hepatocyte inflammation, followed byfibrosis, causing aggravation to hepatic cirrhosis and that, therefore,hepatitis might be suppressed and development into cirrhosis preventedwhen apoptosis is promoted.

The present invention provides an apoptosis regulating compositioncomprising, as an active ingredient, an effective amount of at least oneof the compounds of general formula (1) and salts thereof in combinationwith a pharmacologically acceptable carrier therefor.

The apoptosis regulating composition of this invention can regulate orcontrol apoptosis and, owing to this action, is effective in themedicinal field as an anticancer agent, antiretrovirus agent, andtherapeutic agent for autoimmune diseases, for thrombocytopenia, forAlzheimer's diseases and for various types of hepatitis, tumormetastasis inhibiting agent etc., as mentioned hereinbefore.

In the general formula (1), the benzoyl group which may have loweralkoxy groups as substituents on the phenyl ring includes benzoyl groupshaving 1 to 3 straight-chain or branched C₁₋₆ alkoxy groups substitutingthe phenyl ring, such as benzoyl, 2-methoxybenzoyl, 3-methoxybenzoyl,4-methoxybenzoyl, 2-ethoxybenzoyl, 3-ethoxybenzoyl, 4-ethoxybenzoyl,4-isobutoxybenzoyl, 4-hexyloxybenzoyl, 3,4-dimethoxybenzoyl,3,4-diethoxybenzoyl, 3,4,5-trimethoxybenzoyl, 2,5-dimethoxybenzoyl, andso on.

Of the active ingredient compound (1) according to the invention,6-[4-(3,4-dimethoxy-benzoyl)-1-piperazinyl]-3,4-dihydrocarbostyril ismost preferable.

The compound (1) which are to serve as active ingredients in accordancewith the invention may readily form pharmacologically acceptable saltswith conventional acids. As such acids, there may be mentioned inorganicacids such as sulfuric acid, nitric acid, hydrochloric acid andhydrobromic acid, and organic acids such as acetic acid,p-toluenesulfonic acid, ethanesulfonic acid, oxalic acid, maleic acid,fumaric acid, citric acid, succinic acid and benzoic acid. These saltscan also be used as active ingredient compounds of the presentinvention, just as the free compounds of general formula (1).

The compounds (1) and salts thereof, can be generally formulated intothe per se conventional pharmaceutical preparations. Such preparationsare prepared using the conventional fillers, extenders, binding agents,moistening agents, disintegrating agents, surfactants, lubricants, andthe like diluents or excipients. These pharmaceutical preparations mayhave various dosage forms selected according to the purposes of therapy,and typical examples thereof are tablets, pills, powders, solutions,suspensions, emulsions, granules, capsules, suppositories, injections(solutions, suspensions, etc.), and opthalmic solutions.

For the manufacture of tablets, a wide variety of carriers so far wellknown in this field can be used. Thus, use can be made of, for example,vehicles or excipients such as lactose,. sucrose, sodium chloride,glucose, urea, starch, calcium carbonate, kaolin, crystalline celluloseand silicic acid, binding agents such as water, ethanol, propanol,simple syrup, glucose solution, starch solution, gelatin solution,carboxymethylcellulose, shellac, methylcellulose, potassium phosphateand polyvinylpyrrolidone, disintegrating agents such as dry starch,sodium alginate, powdered agar, powdered laminaran, sodium hydrogencarbonate, calcium carbonate, polyoxyethylene sorbitan fatty acidesters, sodium lauryl sulfate, stearic acid monoglyceride, starch andlactose, disintegration inhibitors such as sucrose, stearin, cacaobutter and hydrogenated oils, absorption promoters such as quaternaryammonium bases and sodium lauryl sulfate, wetting agents or humectantssuch as glycerol and starch, adsorbents such as starch, lactose, kaolin,bentonite and colloidal silica, and lubricants such as refined talc,stearic acid salts, powdered boric acid and polyethylene glycol. Whennecessary, the tablets may further be provided with a conventionalcoating to give, for example, sugar-coated tablets, gelatin-coatedtablets, enteric-coated tablets, film-coated tablets, or double-coatedor multilayer tablets.

For the manufacture of pills, a wide variety of carriers well known inthe art can be used. Examples are vehicles or excipients such asglucose, lactose, starch, cacao butter, hardened vegetable oils, kaolinand talc, binding agents such as powdered gum arabic, powderedtragacanth gum, gelatin and ethanol, and disintegrating agents such aslaminaran and agar.

For the manufacture of suppositories, a wide variety of carriers so farknown can be used. As examples, there may be mentioned polyethyleneglycol, cacao butter, higher alcohols, higher alcohol esters, gelatinand semisynthetic glycerides.

In preparing injections, the solutions or suspensions are preferablysterilized and are preferably isotonic with blood and, for preparingsuch dosage forms, all the diluents in conventional use in this fieldcan be employed. Thus, for example, water, ethyl alcohol, propyleneglycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcoholand polyoxyethylene sorbitan fatty acid esters may be mentioned. In thiscase, the pharmaceutical preparations may contain sodium chloride,glucose or glycerol in an amount sufficient to give isotonic solutions.It is possible to add conventional solubilizing agents, buffers,soothing agents or local anesthetics, etc.

Furthermore, when necessary, the pharmaceutical preparations may containcoloring matters, preservatives, perfumes, flavoring agents, sweeteningagents and the like as well as other drugs.

The proportion of the active ingredient compound in these pharmaceuticalpreparations of this invention is not critical but may suitably beselected in a wide range. Generally, however, the proportion isrecommendably selected within the range of about 1 to about 70% byweight, preferably about 1 to about 30% by weight.

The route of administration of these pharmaceutical preparations of thisinvention is not critical, either, but is selected according to thedosage form, the patient's age, sex and other factors and the severityof the disease to be treated. Thus, for instance, when they are providedin the form of tablets, pills, solutions, suspensions, emulsions,granules or capsules, the preparations are administered orally.Injectable solutions are administered intravenously, either alone or inadmixture with conventional fluids for parenteral infusion containingglucose, amino acids and so on. Where necessary, these solutions mayalso be administered as it is by the intra muscular, intradermal,subcutaneous or intraperitoneal route. Suppositories are administeredrectally. Ophtalmic solutions are drop lotion in the eyes.

While the dosage of the above pharmaceutical preparations is dependenton the method of administration, the patient's age, sex and otherbackground factors, severity of the disease and so on, it is generallyrecommended to administer about 0.5 to 30 mg, as the active ingredient,viz. compound (1), per kilogram body weight per day. The amount of theactive ingredient to be contained in each dosage unit is about 10 to1000 mg.

The diseases against which the apoptosis regulating composition of thepresent invention can be expected to be effective based on the celldifferentiation inducing and other activities thereof include, amongothers, cancer, AIDS, ARC (AIDS related complex), ATL (adult T cellleukemia), hairy cell leukemia, myelopathy (HAM/TSP), respiratorydisorder (HAB/HABA) arthropathy (HAAP), uveitis (HAU), other HTLV-I(human T cell leukemia virus type I) related diseases, autoimmunediseases such as SLE (systemic lupus erythematosus), collagen diseasessuch as rheumatoid arthritis (RA), ulcerative colitis, Sjogren'ssyndrome, primary biliary hepatic cirrhosis, idiopathic thrombocytopenicpurpura (ITP), autoimmune hemolytic anemia, maysthenia gravis,Hashimoto's disease, and insulin dependent (type I) diabetes mellitus.The apoptosis regulating composition of the invention is also applicableto various diseases accompanied by thrombocytopenia, for examplemyelodysplastic syndrome, periodic thrombocytopenia, aplastic anemia,idiopathic thrombocytopenia, disseminated intravascular coagulation,etc. The composition of the invention is further applicable to variousother diseases including various types of hepatitis (such as types C, A,B, and F), Alzheimer's disease, myocarditis, ARDS (adult respiratorydistress syndrome), infectious diseases, liver cirrhosis, prostatichypertrophy, uterine myoma, bronchial asthma, arteriosclerosis, variouscongenital malformations, nephritis, senile cataract, chronic fatiguesyndrome, and myodystrophia.

The apoptosis regulating composition of the present invention, whenadministered as an anticancer composition, for instance, inducesdifferentiation of cancer cells, subsequently promotes or inhibitsapoptosis induction or, directly promotes or inhibits apoptosisinduction, and thereby produces an anticancer effect. In this case, thecomposition of the invention, irrespective of dosage form and/or routeof administration, can be used in combination with one or more ofvarious anticancer agents known as cancer chemotherapeutic agents and/orradiation therapy. The active ingredient compound of the invention whichcan produce an excellent anticancer effect can thus markedly promote theeffect of the other anticancer agent or agents combinedly used, toproduce a synergistic effect. Therefore, even when the partneranticancer agent or agents are used in doses much smaller than the usualdoses, a satisfactory anticancer effect can be obtained, whereby theadverse effects of the partner anticancer agent or agents can beminimized. As such chemotherapeutic agents, there may be mentioned, forexample, 5-fluorouracil (5-FU; Kyowa Hakko Kogyo), mitomycin C (KyowaHakko Kogyo), futraful (FT-207; Taiho Pharmaceutical), endoxan (Shionogi& Co.) and toyomycin (Takeda Chemical Industries).

When used in the treatment of thrombocytopenia, the apoptosis regulatingcomposition of the invention can produce a cell differentiationinduction promoting action and at the same time an apoptosis suppressingaction in patients with MDS such as RA or RARS, thus stimulatingproliferation of hemopoietic cells and causing normal differentiationand maturation. In patients with MDS such as RAEB or RAEB-t,administration of the composition of the invention can result in induceddifferentiation of blast cells and inhibition of blast cellmultiplication, whereby proliferation of mature cells can be caused. Thecomposition of the invention can further be expected to act onpromegakaryocytes and megakaryocytes and promote their differentiationand maturation, thereby promoting thrombopoiesis.

For use in the treatment of thrombocytopenia, the apoptosis regulatingcomposition of the invention can be used in combination with one or moreother known drugs such as thrombopoiesis promoting agents to potentatethese partner drugs. Thus, in some instances, even when the partnerdrugs are used in fairly reduced doses, a satisfactory therapeuticeffect can be produced and the adverse effects of said drugs can bethereby reduced.

The apoptosis regulating composition of the invention is useful also asa therapeutic and prophylactic agent for Alzheimer's disease. In thiscase, in patients with classical Alzheimer's disease or senile dementiaof Alzheimer type, the composition of the invention exhibits an NGF-likeaction through inhibition of apoptosis, thus producing theabove-mentioned therapeutic and prophylactic effects. Further, in thatcase, the composition of the invention can be used in combination withany of the conventional therapeutic agents for Alzheimer's disease suchas cerebral circulation ameliorating agents and cerebral metabolicagents, whereby their effects can be promoted and their adverse effectsreduced in some instances.

The apoptosis regulating composition of the present invention can beused as a cirrhosis preventive agent which controls apoptosis inpatients with drug-induced hepatitis or vital hepatitis to therebymanifest a therapeutic effect in hepatitis and prevent hepatocytes fromfibrogenesis.

Some dosage form examples for the apoptosis regulating composition ofthe invention, and results of pharmaceutical studies on the activeingredient compounds are presented below.

In the following pharmacological test examples, the accompanyingdrawings are referred to, wherein;

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows DNA fragmentation results in the test of apoptosisregulating effect according to the pharmacological test example 1;

FIG. 2 is a graph which shows the results of M12 melanoma cell growthinhibition according to the pharmacological test example 5;

FIG. 3 is a graph showing the results of life-prolonging effect onautoimmune disease obtained by the pharmacological test example 8;

FIGS. 4 to 7 show the results of the thrombocytopenia-improving effectaccording to the pharmacological test example 9;

FIG. 8 shows the results of therapeutic effect on autoimmune diseaseaccording to the pharmacological test example 10;

FIGS. 9 and 10 are photographs showing the effects on PC12 cellsaccording to the pharmacological test example 11;

FIG. 11 is a graph which shows the effect on hepatic failure accordingto the pharmacological test example 12;

FIG. 12 is a graph which shows the effect of B12 melanoma cells onexperimental model of pulmonary metastasis according to pharmacologicaltest example 13.

DOSAGE FORM EXAMPLE 1

    ______________________________________    6-[4-(3,4-Dimethoxybenzoyl)-1-piperazinyl]-                               150    g    3,4-dihydrocarbostyril    Avicel                     40     g    (trademark, Asahi Chemical Industry Co., Ltd.)    Corn starch                30     g    Magnesium stearate         2      g    Hydroxypropylmethylcellulose                               10     g    Polyethylene glycol 6000   3      g    Castor oil                 40     g    Methanol                   40     g    ______________________________________

The above active ingredient compound, Avicel, corn starch and magnesiumstearate are mixed and ground together and the resulting mixture iscompression-molded with a dragee R10 mm punch. The tablets thus obtainedare coated with a film coating composition consisting ofhydroxypropylmethylcellulose, polyethylene glycol 6000, castor oil andmethanol to give film-coated tablets.

Dosage Form Example 2

    ______________________________________    6-[4-(3,4-Dimethoxybenzoyl)-1-piperazinyl]-                              150.0  g    3,4-dihydrocarbostyril    Citric acid               1.0    g    Lactose                   33.5   g    Dicalcium phosphate       70.0   g    Pluronic F-68             30.0   g    Sodium lauryl sulfate     15.0   g    Polyvinylpyrrolidone      15.0   g    Polyethylene glycol (Carbowax 1500)                              4.5    g    Polyethylene glycol (Carbowax 6000)                              45.0   g    Corn starch               30.0   g    Dry sodium lauryl sulfate 3.0    g    Dry magnesium stearate    3.0    g    Ethanol                   q.s.    ______________________________________

The above active ingredient compound, citric acid, lactose, dicalciumphosphate, pluronic F-68 and sodium lauryl sulfate are admixed.

After size selection using a No. 60 screen, the mixture is granulated bythe wet process using an alcoholic solution containingpolyvinylpyrrolidone, Carbowax 1500 and Carbowax 6000. When necessary,alcohol is added to make the powder into a paste-like mass. Then, cornstarch is added and the blending is continued until uniform granules areformed. The mixture is then passed through a No. 10 screen, placed in atray and dried in an oven maintained at 100° C. for 12 to 14 hours. Thedried granules are sieved through a No. 16 screen, then dry sodiumlauryl sulfate and dry magnesium stearate are added and, after blending,the mixture is compressed to a desired size and shape using a tabletingmachine.

The above cores are treated with a varnish and dusted with talc forpreventing absorption of moisture and then provided with an undercoatlayer. Varnish coating is repeated as many times as sufficient forinternal use. The tablets are rendered completely round and smooth byapplication of a further undercoat layer and a smooth coating. Coloringcoating is conducted until a desired coloring is obtained. After drying,the coated tablets are polished to give uniformly polished tablets.

Pharmacological tests, described hereinafter, were performed using thefollowing test compounds.

1. 6-[4-(3,4-Dimethoxybenzoyl)-1-piperazinyl]-3,4-dihydrocarbostyril

2. 6-[4-(3,4,5-Trimethoxybenzoyl)-1-piperazinyl]-3,4-dihydrocarbostyril

3. 7-[4-(3,4,5-Trimethoxybenzoyl)-1-piperazinyl]-3,4-dihydrocarbostyril

4. 7-[4-(3,4-Dimethoxybenzoyl)-1-piperazinyl]-3,4-dihydrocarbostyril

5. 6-[4-(4-Ethoxybenzoyl)-1-piperazinyl]-3,4-dihydrocarbostyril

6. 5-[4-(3,4-Dimethoxybenzoyl)-1-piperazinyl]-3,4-dihydrocarbostyril

7. 6-[4-(3,4-Dimethoxybenzoyl)-1-piperazinyl]carbostyril

8. 8-[4-(3,4-Dimethoxybenzoyl)-1-piperazinyl]-3,4-dihydrocarbostyril

Pharmacological Test Example 1 Apoptosis-regulating effect

CMK cells were used to prepare a suspension of 1×10⁵ cells/ml inRPMI-1640 medium supplemented with 10% fetal calf serum and Compound 1was added at a concentration of 30 μg/ml. The mixture was incubated in aculture flask at 37° C. for 2 or 4 days. As a control, the medium towhich the solvent alone was added was similarly incubated.

Cells were harvested by centrifugation at 4° C. for 10 minutes at 200 xG. The pellet was dissolved in a hypotonic buffer (10 mM Tris-HCl, 1 mMEDTA and 0.2% Triton X 100, pH 7.5) and the solution was centrifuged at4° C. for 10 minutes at 13000 x G. The supernatant was collected andallowed to stand in 50% isopropyl alcohol -0.5M NaCl at -20° C.overnight. The resultant precipitate was centrifuged at 4° C. for 10minutes at 13000 x G, the supernatant was discarded, and the sedimentwas dried. The dry sediment was resuspended in 10 mM Tris-HCl-1 mM EDTA(pH 7.4). To the supernatant was added RNase at a final concentration of0.1 mg/ml and the mixture was incubated at 37° C. for 1 hour.

To a sample corresponding to the cell count of 1×10⁶ was added 1/5volume of a loading buffer containing 15 mM EDTA, 2% SDS, 50% glyceroland 0.05% bromphenol blue, and the mixture was incubated at 65° C. for10 minutes.

The culture sample thus obtained was subjected to electrophoresis in1.5% agarose gel at 100 V for 100 minutes. The DNA was stained withethidium bromide.

The results are presented in FIG. 1.

In the figure, lanes at both sides represent markers 1 (120, 600 and1350 bp) and 2 (72, 420 and 930 bp), respectively. The second lane fromthe left shows the pattern after 2 days of treatment with Compound 1,the third lane from the left the pattern after 2 days of controltreatment, the fourth lane from the left the pattern after 4 days oftreatment with Compound 1, and the fifth lane from the left the patternafter 4 days of control treatment.

It is, thus, clear that the expression of DNA fragments appearstime-dependently on the electrophoregram. This indicates that Compound 1promotes apoptosis of CMK cells.

Pharmacological Test Example 2 Cancer cell growth inhibition

(1) Human promyelocytic leukemia cells (HL-60)

The human promyelocytic leukemia cell line HL60 is a human leukemia cellline established by Robert Gallo et al. and typical properties thereofare described in the literature [Gallo, R. C. et al., Blood, 54, 713(1979)]. It is available from the American Type Culture Collection(ATCC) under the accession number ATCC No. CCL-240.

The concentration of HL-60 cells was adjusted to 1×10⁵ cells/ml usingRPMI-1640 medium (GIBCO) supplemented with 10% of FCS (fetal calf serum;GIBCO).

(2) Human stomach cancer cells (KATO-III)

The cell concentration was adjusted to 1×10⁵ cells/ml using the samemedium as used above in (1).

(3) Test compound solutions

Test compound 1 was dissolved in acetic acid, followed by dilution withthe same medium as used above in (1) and neutralization with 2N NaOH togive final concentrations of 100 μg/ml, 10 μg/ml and 1.0 μg/ml.

(4) Giemsa stain solution

Giemsa stain solution was prepared by diluting Giemsa reagent (Merck)50-100 times with phosphate buffer (pH 6.4).

(5) Esterase staining reagent

A reagent for esterase staining (Muto Kagaku) was used according to thenaphthol AS-D chloroacetate method.

(6) Anti-CD33 monoclonal antibody My9

The monoclonal antibody My9 (Coulter) capable of specifically reactingwith immature granulocytes was dissolved by adding 0.5 ml of distilledwater per vial.

(7) Results

a) The test compound solutions prepared as described above under (3)were added to the HL-60 cell suspension prepared as described aboveunder (1) to final concentrations of 10 μg/ml and 1.0 μg/ml. Using12-well culture plates (Costar), cultivation was conducted in a 5%carbon dioxide gas incubator maintained at 37° C. for 3 to 4 days. Thetest compound-free medium was used as a control. Thereafter, the cellsuspension in each well was sampled and mixed with 0.2% trypanblue-containing phosphate buffer, and unstained viable cells werecounted under a microscope. Based on the counting result, the viablecell concentration was readjusted to 1×10⁵ cells/ml, and cultivation wascontinued under the same conditions.

The cell growth inhibiting effects on day 27 and day 33 are shown inTable 1.

KATO-III cells prepared as described above under (2) were cultivated inthe same manner. The cell growth inhibiting effects on day 24 and day 33are shown in Table 2.

                  TABLE 1    ______________________________________                  Cell growth (%)                              Cell growth (%)    Test compound 1                  On day 27   On day 33    ______________________________________    0 (Control)   100         100    1.0 μg/ml  76 ± 3.8 0    10 μg/ml   54 ± 2.6 0    ______________________________________

                  TABLE 2    ______________________________________                  Cell growth (%)                              Cell growth (%)    Test compound 1                  On day 24   On day 33    ______________________________________    0 (Control)   100         100    1.0 μg/ml  88 ± 2.3 0    10 μg/ml   61 ± 1.5 0    ______________________________________

From the data shown in Table 1 and Table 2, it is apparent that thegrowth of tumor cells is inhibited in a manner dependent on theconcentration of the test compound.

b) Differentiation of HL-60 cells

HL-60 cells prepared as described above under (1) were cultivated in thesame manner as in a). Cells collected on day 27 were placed on a slideglass and subjected to Giemsa staining, followed by morphologicalobservation under a microscope. When the test compound was added,differentiation into cells with granule was observed as a morphologicalchange as contrasted with the test compound-free case. The results ofesterase staining by the naphthol AS-D chloroacetate method indicateddifferentiation of 70-85% of HL-60 cells into granulocytes.

c) Reactivity with anti CD33 monoclonal antibody My9

HL-60 cells prepared as described above under (1) were cultivated in thesame manner as in a). On day 27, the cell concentration was adjusted to1×10⁶ cells/ml and 100 μl of each aliquot was reacted with 10 μl offluorescein (FITC)-labeled My9, followed by flow cytometry. The positiverate data thus obtained are shown in Table 3.

                  TABLE 3    ______________________________________    Test Compound 1                   Positive rate (%)    ______________________________________    0 (Control)    97    1.0 μg/ml   90    10 μg/ml    77    ______________________________________

From the data shown in Table 3, it was noted that when the test compoundwas added, the proportion of undifferentiated HL-60 cells decreasedwhile the proportion of differentiated cells increased.

The above results indicated that the active ingredient compound of thepresent invention has growth inhibiting and differentiation inducingeffects on tumor cells.

Pharmacological Test Example 3 Differentiation inducing test

(8) Monoclonal antibody FH-6

The monoclonal antibody FH-6 capable of recognizing sialyl Le^(X) wasfluoresein (FITC)-labeled, and the reactivity between the activeingredient compound of the present invention and FH-6 was examined byflow cytometry as follows.

(9) Reactivity with monoclonal antibody FH-6

The test compound solutions prepared as described above under (3) wereadded to HL-60 cells prepared as described above under (1) to a finalconcentration of 1.0 μg/ml or 10 μg/ml. Cultivation was conducted in a5% carbon dioxide gas incubator at 37° C. for 2 hours. Thereafter, thecell concentration was adjusted to 1×10⁶ cells/ml and 100 μl of eachaliquot was reacted with 10 μl of the fluorescein-labeled FH-6 solution,followed by flow cytometry. The positive rate data thus obtained areshown in Table 4.

                  TABLE 4    ______________________________________    Test Compound 1                   Positive rate (%)    ______________________________________    0 (Control)    95    1.0 μg/ml   82    10 μg/ml    76    ______________________________________

Form the above results, it was revealed that the addition of the activeingredient compound of the present invention resulted in loss of sialicacid from the HL-60 cell surface. This means differentiation of HL-60cells and increase in sialidase activity.

Pharmacological Test Example 4 Effect on human promyelocytic leukemiacells (HL-60)

HL-60 cells were cultivated in RPMI-1640 medium supplemented with 10% ofFCS at 37° C. under 5% CO₂, and the cell concentration was adjusted to5×10⁴ cells/ml.

Then, the above-mentioned medium containing 30 μg/ml of each testcompound was added to each well of 6-well microplates (Costar), followedby 3 days of incubation at 37° C. under 5% CO₂. In a control group, theabove medium alone was added and incubation was performed in the samemanner. Thereafter, each cultured cell suspension was sampled into anEppendorf tube and, after staining with 0.2% trypan blue-containingphosphate buffer, viable cells were counted using a hematocytometer.

The above cells were adjusted to 1×10⁷ cells/ml. To 100 μl of the cellsuspension was added 5 μl of a solution of fluorescein isothiocyanate(FITC)-labeled anti-human CDllb antibody (Mol; Coulter) and the reactionwas allowed to proceed on ice in the dark for 30 minutes. Then, cellswere washed twice with PBS (phosphate-buffered saline; NissuiPharmaceutical) containing 0.1% of BSA (bovine serum albumin; Sigma),finally suspended in 500 μl of the BSA-containing PBS and measured forfluorescence intensity by flow cytometry using Profile II (Coulter).

The results thus obtained are shown in Table 5 and Table 6.

                  TABLE 5    ______________________________________                 Induction of differentiation    Test compound                 promoting effect    ______________________________________    1            322    3            384    4            383    5            232    6            303    7            898    8            344    ______________________________________

                  TABLE 6    ______________________________________                  Cell growth    Test compound inhibiting effect (%)    ______________________________________    2             20.2    7             24.1    8             24.9    ______________________________________

From the data shown in the above tables, it was noted that, in each testcompound group, a cell growth inhibiting effect was produced while theexpression of CDllb was increased whereby the induction ofdifferentiation into the granulocyte, monocyte and macrophage series waspromoted.

Pharmacological Test Example 5 M12 melanoma cell growth inhibition

This study was performed in groups of 10 Balb/c nude mice.

On the first day of the experiment (day 0), 2×10⁶ M12 melanoma cellswere transplanted into mice in the test group. When the tumor volumereached 100 mm³, the test Compound 1 was administered orally in doses of10 mg/kg daily to the mice. This treatment was carried out 30 times fromday 25 to day 55. The tumor diameter was measured using caliperseveryday after transplantation of tumor cells. The tumor volume wascalculated according to the following formula.

    Tumor volume (mm.sup.3)=(long diameter)×(short diameter).sup.2 ×1/2

The results are shown in FIG. 2.

In FIG. 2, the corresponding results in the control group are alsopresented. In the figure, * indicates a significant difference at p<0.05(Student's t test).

As seen from FIG. 2, the administration of the test compoundsignificantly inhibits growth of M12 melanoma cells.

Phamacological Test Example 6 ATL-infected cell growth inhibition

The following pharmacological test was performed using Compound 1 as thetest compound.

(a) Preparation of human peripheral blood acute lymphocytic leukemiacells (Molt-4)

Human peripheral blood acute lymphocytic leukemia cells (Molt-4) [J.Minowadat et el., J. Natl. Cancer Inst., 49, 891-895 (1972): ATCCCRL1582] were used to prepare a suspension of 1×10⁵ cells/ml inRPMI-1640 medium (Gibco) supplemented 10% fetal calf serum (FCS, Gibco)for use as ATL virus-noninfected cells.

(b) Preparation of human adult T cell leukemic cells (5S)

Human adult T cell leukemia cells (5S) were used to prepare a suspensionof 1×10⁵ cells/ml in RPMI-1640 medium (Gibco) supplemented 10% fetalcalf serum (FCS, Gibco) for use as ATL virus-infected cells.

(c) Preparation of the test compound

Compound 1 was dissolved in hydrochloric acid, diluted with the samemedium as above, neutralized with 2N NaOH and adjusted to concentrationsof 1 μg/ml, 10 μg/ml and 30 μg/ml.

(d) Pharmacological test

The cells prepared in (a) and (b) above were respectively placed inwells of a 24-well culture plate (Nunc), followed by addition of thetest compound prepared in (c) (final concentration: 1 μg/ml, 10 μg/ml or30 μg/ml), and cultured at 37° C. in a 5% carbon dioxide incubator for 4days. As a control, the medium without addition of the test compound wasused. After completion of incubation, the cell suspension in each wellwas taken for determination of viable cells.

The results are shown in Table 7.

                  TABLE 7    ______________________________________    Compound 1 (μg/ml)                     0      1        10   30    ______________________________________    Nummber of ATLV-noninfected                     13.9   13.9     12.2 12.8    cells (× 10.sup.5)    Number of ATLV-infected                      6.5    0.6      0.2  3.3    cells (× 10.sup.5)    ______________________________________

As seen from Table 7, the active ingredient compound of the invention(Compound 1) exerts a potent inhibitory effect on the growth ofATLV-infected cells at a concentration of at least 1 μg/ml, while itdoes not affect ATLV-noninfected cells.

Pharmacological Test Example 7 Antiretrovirus activity test

(a) Preparation of human transformed normal T cells (MT-4)

Human transformed normal T cells (MT-4) [Nagumo, T. and Hoshino, H.,Jpn. J. Cancer Res. 79, 9-11 (1988)] were used to prepare a suspensionof 2×10⁵ cells/ml in RPMI-1640 medium (Gibco) supplemented 10% FCS(Gibco).

(b) Preparation of the test compound

Compound 1 was dissolved in hydrochloric acid, diluted in the samemedium as above, neutralized with 2N NaOH and adjusted to concentrationsof 1 μg/ml, 10 μg/ml, 30 μg/ml and 100 μg/ml.

(c) Phamacological test

The cells prepared in (a) above and the test compound (finalconcentration: 1 μg/ml, 10 μg/ml, 30 μg/ml or 100 μg/ml) prepared in (b)above were placed in the wells of a 12-well culture plate (Costar).Then, 50 μl/well of HIV suspension was added to infect the cells in eachwell, followed by 3-day incubation at 37° C. in a 5% carbon dioxideincubator. As a control, the medium without addition of the testcompound was used. After completion of incubation, the cell suspensionin each well was taken and viable cells were counted.

The cell suspension in each well was then centrifuged (1500 rpm×5 min)and 750 μl of culture supernatant was obtained.

The culture supernatant, 750 μl, obtained above was placed in anEppendorf tube (1.8 ml) and 4M NaCl and PEG-6000 (Sigma) were added. Themixture was allowed to react at 0° C. for 2 hours to precipitate thevirus and the virus was harvested by centrifugation (15000 rpm×20 min).To this virus pellet were added solution I [50% glycerol (Wako PureChemical), 25 mM Tris-HCl (pH 7.5), 50 mM KCl, 0.025% Triton X-100(Sigma) and 5 mM DTT (Sigma)] and solution II [0.9% Triton X-100 (Sigma)and 1.5M KCl] for lysis at 0° C. for 30 min. To the resultant 10-μlvirus solution were added 5 mg/ml bovine serum albumin (BSA, Sigma), 1MTris-HCl, 100 mM DTT (Sigma), 1M KCl, 10 mM Poly A (Sigma), 0.15 mM TTP(Sigma), 200 mM MgCl₂, 0.15 mM Oligo(dT) (NEN) and [³ H]-TTP (NEN) andthe mixture was allowed to react at 37° C. for 1 hour. The reaction wasthen stopped by placing the mixture in ice and the whole amount wasspotted on a membrane filter (DE-81 Filter, Whatman) and dried. Afterdrying, the filter was washed three times with 0.5M Na₂ HPO₄ and rinsedin ethanol and dried. The radioactivity of [³ H]-TTP taken into DNA onthe membrane was counted with a liquid scintillation counter todetermine reverse transcriptase activity (unit, cpm).

The viable cell count (×10⁴ cells) and reverse transcriptase activity(cpm) found as above are shown in Table 8.

                  TABLE 8    ______________________________________    Compound 1 (μg/ml)                0        1      10     30   100    ______________________________________    Number of HIV-                 105      100    88     78   85    infected cells    (× 10.sup.4)    Reverse transcriptase                1316     1423   1494   372  898    activity (cpm)    ______________________________________

From Table 8, it was revealed that the active ingredient compound ofthis invention (Compound 1) not only inhibits cell growth but inhibitsreverse transcriptase activity remarkably at doses of 30 μg/ml and 100μg/ml.

The results of the above pharmacological test examples 6 and 7demonstrate that the compound used as the active ingredient of thisinvention has high anti-retrovirus activity.

Pharmacological Test Example 8 Effect of the active ingredient compoundof this invention in an animal model of autoimmune disease

W/BF₁ :(NZW×BXSB)F₁ mice are mice with spontaneous autoimmune disease.They are known as an animal model of lupus nephritis accompanied, withhigh frequency, by hypertension and myocardial infarction [Hang, L. M.,et al., J. Exp. Med., 154, 216-221 (1981)]. They develop anti-plateletantibodies with increasing age, showing marked thrombocytopenia, and areused as an animal model of idiopathic thrombocytopenic purpura (ITP) aswell [Oyaizu, N., et al., J. Exp. Med. 167, 2017-2077 (1988)]. It hasbeen reported that more than 90% of the model animals die of myocardialinfarction or renal failure within 8 months of birth [Ikehara, S.,Metabolism and Disease, (Suppl) 26, 169-176 (1989)].

The life-prolonging effect of Compound 1 of this invention and theeffects of the same compound on blood cells and anti-platelet antibodieswere investigated in these W/BF₁ :(NZW×BXSB)F₁ mice.

W/BF₁ :(NZW×BXSB)F₁ mice were used at the age of 14 weeks (purchasedfrom Kiwa Experimental Animal). Groups of 10 mice were used in eachexperiment.

The mice in respective groups were given a diet (Oriental Yeast), whichis usually given to this kind of mice, and water.

Compound 1 (Lot No. 9D87M) was used as suspended in a 0.5% solution ofcarboxymethylcellulose (CMC, Cellogen). Compound 1 was administered tomice by oral garage in doses of 100 and 300 mg/kg bodyweight/day for 6weeks from 14 weeks of age to 20 weeks of age according to the 5-dayadministration and 2-day cessation schedule. As a control, an untreatedgroup was provided.

(1) For investigation of the life-prolonging effect, the animals wereobserved for survival or death from 14 to 20 weeks of age. Statisticanalysis was performed by the generalized Wilcoxon test with respect tothe untreated group. The results are shown in FIG. 3.

In the figure, the ordinate represents the survival rate (%) and theabscissa represents the age in weeks (14 weeks to 20 weeks).

As seen from the figure in the group given 100 mg/kg/day of Compound 1of this invention, no death occurred till the age of 20 weeks. In the300 mg/kg/day group, two animals were found dead at 18 weeks of age. Inthe untreated group, two, one and three animals were found dead at 17,18 and 19 weeks of age, respectively. Thus, the life-prolonging effectof the active ingredient compound of this invention (Compound 1) in amouse model of autoimmune disease was confirmed.

(2) For blood cell analysis, the blood was sampled from the fundus oculiat the age of 14, 16, 18 and 20 weeks. Changes in blood cell (whiteblood cell, red blood cell and platelet) counts were determined using anautomatic blood cell analyzer (Coulter JR, Coulter). The differential(neutrophil and lymphocyte) counts were determined using blood smearsstained with Wright-Giemsa stain. For statistical analysis, Student's ttest was used, taking the untreated group as control, on each bloodsampling day.

The results are shown in Table 9.

                                      TABLE 9    __________________________________________________________________________                       Weeks of age            Treatment  14      16      18     20    __________________________________________________________________________    White blood cells            Untreated  8.23 ± 0.99                               15.34 ± 4.53                                       10.87 ± 1.76                                              13.30 ± 4.04    (× 10.sup.3 /mm.sup.3)            Compound 1                  100 mg/kg                       7.53 ± 1.26                               12.91 ± 4.76                                       16.49 ± 4.98                                              11.72 ± 1.71                  300 mg/kg                       5.46 ± 0.74                               9.36 ± 1.77                                       11.90 ± 3.52                                              12.97 ± 5.95    Neutrophils            Untreated  2.06 ± 0.44                               3.25 ± 1.90                                       2.38 ± 0.63                                              3.84 ± 1.86    (× 10.sup.3 /mm.sup.3)            Compound 1                  100 mg/kg                       0.97 ± 0.21                               3.01 ± 2.14                                       5.82 ± 1.65                                              3.25 ± 1.06                  300 mg/kg                       0.79 ± 0.19                               1.86 ± 0.40                                       5.65 ± 2.77                                              2.24 ± 0.23    Lymphocytes            Untreated  6.17 ± 0.92                               12.06 ± 2.68                                       8.49 ± 1.39                                              9.47 ± 2.23    (× 10.sup.3 /mm.sup.3)            Compound 1                  100 mg/kg                       6.55 ± 1.11                               9.83 ± 2.72                                       10.68 ± 3.78                                              8.70 ± 1.02                  300 mg/kg                       4.50 ± 0.63                               7.01 ± 0.92                                       6.16 ± 0.88                                              10.73 ± 5.81    Red blood cells            Untreated  9.12 ± 0.53                               7.82 ± 0.44                                       8.00 ± 0.60                                              6.80 ± 0.53    (× 10.sup.6 /mm.sup.3)            Compound 1                  100 mg/kg                       9.08 ± 0.19                                9.06 ± 0.19*                                       8.46 ± 0.34                                               8.29 ± 0.37*                  300 mg/kg                       9.17 ± 0.21                               8.43 ± 0.30                                       8.83 ± 0.32                                              7.90 ± 0.30    Platelets            Untreated  1144.5 ± 121.3                               781.3 ± 115.5                                       670.3 ± 123.8                                              228.0 ± 90.0    (× 10.sup.3 /mm.sup.3)            Compound 1                  100 mg/kg                       1225.2 ± 187.6                               749.6 ± 116.6                                       524.0 ± 105.6                                              341.1 ± 111.9                  300 mg/kg                       1316.8 ± 86.3                               1055.8 ± 72.0                                       980.0 ± 114.4                                               823.3 ± 24.5**    __________________________________________________________________________     *: P<0.05     **: P<0.01

As described in the above-mentioned report of Ikehara, the hematologicalfeature of W/BF₁ mice is development of leukocytosis andthrombocytopenia with increasing age. In both the untreated group andthe group given Compound 1 of this invention, the white blood cell counttended to increase with age, indicating that the compound of thisinvention has no effect on this parameter. The platelet count in theuntreated control group began to decrease at 14 weeks of age(1144.5±121.3/mm³) and, at 20 weeks of age, decreased to about one fifth(228.0±90.0/mm³) the count found at 14 weeks of age. However, in thegroup given 300 mg/kg/day of the active ingredient compound of thisinvention (Compound 1), inhibition of decrease in platelet count beganto appear immediately after initiation of treatment and a significantinhibition was found as compared to the untreated group (228.0±90.0/mm³)at 20 weeks of age (823.3±24.5/mm³). A similar result was found for redblood cell count.

(3) Oyaizu et al. have reported that in W/BF₁ mice, thrombocytopenia isassociated with the appearance of anti-platelet antibodies. Since, asmentioned above in (2), the administration of Compound 1 of theinvention inhibited a decrease in platelet count, the assay ofanti-platelet antibodies was carried out at the age of 20 weeks.

The platelets collected from Balb/c mice and the plasma collected fromW/BF₁ mice treated up to the age of 20 weeks were incubated together atroom temperature for 30 minutes. The mixture was washed twice andallowed to react with FITC-labeled anti-mouse IgG (Tago, Code No. 6250),and after staining, anti-platelet antibodies were assayed with an FACSanalyzer (EPICS-Profile II, Coulter). The negative control was set sothat the plasma from Balb/c mice was 98% negative. The positive rate ofeach sample was determined.

The results are shown in Table 10.

                  TABLE 10    ______________________________________    (% positive)                         Compound 1                                   Compound 1    Mouse No.            Untreated    100 mg/kg 300 mg/kg    ______________________________________    1       N.T           3.7       2.3    2       20.9         13.5      11.4    3       N.T           3.1      14.1    4       N.T          17.3       6.0    5       N.T          21.5      N.T    6       26.0         16.8      16.4    7       N.T          18.2      12.1    8       27.9          7.8       4.1    9        3.7          8.5      19.5    10      N.T          27.1      N.T    (Mean ± SE)            19.6 ± 5.5                         13.3 ± 2.7                                   10.7 ± 2.2    ______________________________________

In the table, N.T. means "not tested" because of death at the time ofdetermination. The numerical figures represent the positive rates (%).

The table shows that whereas the positive rate in the untreated groupwas 19.6±5.5%, the positive rate was 13.2±2.7% in the group given 100mg/kg/day of the active ingredient compound of this invention(Compound 1) and 10.7±2.2% in the 300 mg/kg/day group, indicating adose-dependent inhibition of anti-platelet antibodies.

As mentioned above, the W/BF₁ mouse is an animal model of spontaneousITP for human chronic ITP. These animals develop lupus nephritis at orafter 3 months of birth and more than 90% of them die of myocardialinfarction or renal failure before 8 months of birth. It has beenreported that these mice can be treated by transplanting bone marrowfrom normal Balb/c mice [Ikehara, S., et al., Proc. Natl. Acad. Sci.U.S.A., 82, 2483-2487 (1985); Yasumizu, R., et al., Proc. Natl. Acad.Sci. U.S.A., 84, 6555-6557 (1987); Ikehara, S., et al., Proc. Natl.Acad. Sci, U.S.A., 56, 3306-3310 (1989)]. In clinical practice, too, themost effective treatment modality available today for ITP is consideredto be bone marrow transplantation.

As seen from the results of above (1)-(3), the active ingredientcompound of this invention exhibits a significant life-prolonging effectin W/BF₁ mice and inhibits thrombocytopenia due to appearance ofanti-platelet antibodies, which is a hematological feature of thisstrain of mice, indicating that the compound has an autoimmunedisease-improving effect.

Pharmacological Test Example 9 Thrombocytopenia-improving effect

1) Preparation of the test compound

Compound 1 was dissolved in 1N hydrochloric acid and diluted with fetalcalf serum (FCS, Gibco) to give a 1 mg/ml solution. This solution wasadded to RPMI-1640 medium (Flow Laboratories) supplemented 10% FCS andthe mixture was neutralized with 1N NaOH and adjusted to a concentrationof 30 μg/ml before use.

2) Preparation of CMK cells

CMK cells were established by T. Sato et al. from patients with acutemegakaryocytic leukemia. They are known to react with anti-plateletantibodies (anti-glycoprotein IIb/IIIa antibody, anti-glycoprotein Ibantibody and Plt-1 antibody). Moreover, it has mega-karyocyte-likeproperties, such as formation of α granules and a demarcation membranein the cytoplasm. Therefore, these cells have been used for the analysisof the growth and differentiation of megakaryocytes [Sato, T., et al.,Exp. Hematol., 15, 495 (1987); Sunami, S., et al., Blood, 70, 368(1987); Komatsu, N., et al., Blood, 74(1), 42-48 (1989); Fuse, A., etal., British J. Haematology, 77, 32-36 (1991); Acta HaematologiaJaponica 53(2), 294-295, 317-318 (1990)].

CMK cells were supplied by Dr. Takeyuki Sato at the Department ofPediatrics, Chiba University School of Medicine. The cells, 5×10⁴cells/ml, were cultured in RPMI-1640 medium supplemented 10% FCS andsub-cultured every four days.

3) Effect of Compound 1 on the expression of platelet-associated antigenof CMK cells

CMK cells, 1×10⁵ cells/ml, were incubated in 10% FCS-added RPMI-1640medium, containing 30 μg/ml of Compound 1, for 4 days and the expressionof platelet-associated antigen on the cell surface was determined byflow cytometry (Coulter) using FITC-labeled Plt-1 antibody (Coulter).The medium containing no Compound 1 was used as a solvent control.

For the analysis of cell surface antigen, 1×10⁶ cells/100 μl of CFLKcells were taken into a tube (Fischer) and reacted with 5 μl ofFITC-labeled Plt-1 antibody at 4° C. for 30 minutes. The reactionmixture was washed three times with phosphate buffer containing 0.1%bovine serum albumin and determined for the percentage positive cellsand fluorescence intensity.

The above-mentioned Plt-1 antibody (Coulter) is an antibody recognizingthe platelet-associated antigen, glycoprotein IIb/IIIa and has beenreported to increase with differentiation or maturation of CMK cells[Komatsu, N., et al., Blood, 74(1), 42-48 (1989)].

The results are shown in Table 11.

                  TABLE 11    ______________________________________                   Plt - 1                     Percentage                               Mean    Cell count       positive  fluorescence    (10.sup.5 /ml)   cells (%) intensity (%)    ______________________________________    Solvent 5.8          96.1      100.0    control    Compound 1            3.2          96.5      191.6    (30 μg/ml)    ______________________________________

The table shows that about 96% of CMK cells used are cells positive forthe platelet-associated antigen recognized by Plt-1 antibody. Theaddition of 30 μg/ml of Compound 1 caused no change in the percentage ofpositive cells.

However, analysis of mean fluorescein intensity, which represents theamount of antigen expression per cell, shows that, with the meanfluorescein intensity of solvent control cells being taken as 100%, theintensity of the cells treated with 30 μg/ml of Compound 1 was about191%. It is, thus, clear that addition of Compound 1 resulted in a2-fold increase in the expression of platelet-associated antigen,recognized by Plt-1 antibody, on the surface of CMK cells.

4) Morphological observation of CMK cells

CMK cells, 1×10⁵ cells/ml, were incubated in 10% FCS-added RPMI-1640medium containing Compound 1 for 4 days and observed for the morphologyof cells under the phase contrast microscope. A portion of the cellpopulation was taken and a cytospin preparation was prepared and stainedwith Wright-Giemsa stain for determination of the cell morphology.

The results are presented in FIGS. 4-7. Thus, FIGS. 4 and 6 are thephase constrast micrograph and stained map, respectively, of the mediumcontrol, and FIGS. 5 and 7 represent the corresponding figures for cellstreated with 30 μg/ml of Compound 1.

In the cells treated with 30 μg/ml of Compound 1 of this invention, thephase contrast micrograph reveals the fragmentation of cells like in theprocess of platelet production. The stained map shows budding of thecell membrane and lobulation of the nuclei in the cells treated withCompound 1 of this invention.

The foregoing results show that the carbostyril derivative, the activeingredient of this invention, increases the expression ofplatelet-associated antigen on the surface of CMK cells, causesfragmentation of cells like in the process of platelet production andfurther causes budding of the cell membrane and lobulation of the nucleiof cells and suggest that said compound promotes the differentiation ormaturation of CMK cells. It is, therefore, expected that Compound 1 actson promegakaryocytes or megakaryocytes to promote their differentiationand maturation by virtue of these characteristic actions and therebystimulate platelet production and alleviate thrombocytopenia.

Pharmacological Example Test 10 Therapeutic effect on autoimmune disease

Groups of five female MRL/Mp-lpr/lpr mice (purchased at 7 weeks of agefrom Charles River Japan, Inc.) were used at the age of 19 weeks.

Mice of the above strain are known to spontaneously develop autoimmunediseases such as human systemic lupus erythematosus (SLE)-like immunecomplex glomerulonephritis, lymphoma, vasculitis and polyarthritis(Theofilopolos, A. and Dixon, F. J., Adv. Immunology, 37, 269 (1985);Murphy, E. D., Roths, J. B., Autoimmunity and lymphoproliferation,Elsevier North Holland, New York, 207-221 (1978); Taniguchi, Y., GendaiIryo, 21, 131 (1989); Abe, C.).

Mice in both the Compound 1-treated group and the control group(untreated group) were given a mouse diet (Oriental Yeast) and water.

Compound 1 was used as suspended in 0-5% carboxymethylcellulose (CMC,Dai-ichi Kogyo Seiyaku Co., Ltd.) solution.

Compound 1 was administered by oral gavage in a dose of 300 mg/kg bodyweight daily from the age of 19 weeks to the age of 25 weeks accordingto the 5-day administration and 2-day cessation schedule (Compound1-treated group).

Mice in the respective groups were examined for changes in urinaryprotein, lymphoma diameter, vasculitis of the auricle, and loss of hair.

As a result, the Compound 1-treated group showed a tendency ofinhibition of the increase in urinary protein as compared with thecontrol group. As to lymphoma size, the diameter in the Compound1-treated group was 7.8±1.3 mm vs. 9.0±0.8 mm in the control group,indicating that the enlargement of lymphoma was slightly inhibited.

FIG. 8 compares the degrees of vasculitis, hair loss and lymphomabetween the groups.

The figure shows improvements in vasculitis and loss of hair andreduction in lymphoma in the Compound 1-treated group as compared to thecontrol group.

The forgoing results indicate that administration of Compound 1 can beexpected to ameliorate autoimmune disease on the basis of itsapoptosis-modifying action.

Pharmacological Test Example 11 Effect on PC12 cells

PC12 cells subcultured in Dulbecco's modified Eagle's medium (D-MEM)supplemented with 5% heat-inactivated (56° C., 30 minutes) horse serumand 10% fetal calf serum (FCS) were transferred onto collagen-coatedplastic Petri dishes (35 mm in diameter) at a concentration of 6×10⁴cells/3 ml medium. On day 2 of transfer, the culture medium was replacedwith D-MEM supplemented with various concentrations of Compound 1, nervegrowth factor (NGF, Wako Pure Chemical) or FCS (control) and culture wasfurther continued. The cells were observed for morphological changesunder the phase contrast microscope on day 3.

The results are shown in FIGS. 9 and 10.

In FIG. 9, the top panels represent the control group in which FCS alonewas added and the bottom panels represent the Compound 1 30 μg/ml group.As seen from the figure, the typical morphological change, i.e.formation of nerve fiber-like processes, is found in the bottom panelsas contrasted to the top panels and the most prominent formation ofnerve fiber-like processes is observed in the presence of FCS 0-3%(bottom, left). When the concentration of FCS was increased, cells ofdifferent shapes appeared (bottom, middle and right). Thus,morphological changes were induced in 30-50% of the total cellpopulation.

In FIG. 10, the top panels represent the NGF alone group (0.3% FCS wasadded, however) and the bottom panels represent the Compound 1 30 μg/mlgroup. This figure shows that like the formation of nerve-like processesdue to NGF stimulation as seen in the top panels, the formation ofnerve-like processes is observed in the Compound 1 group as well. Thebottom panels show that the combination of Compound 1 and NGF (however,in the presence of 0.3% FCS) promotes elongation of the process ascompared with Compound 1 or NGF alone.

Pharmacological Test Example 12 Effect on hepatic failure

Heat-killed cells of P. acnes (a gift from Dr. Mizoguchi, the ThirdDepartment of Internal Medicine, Osaka City University School ofMedicine) were administered intravenously in a dose of 3 mg/body to maleBalb/c mice (7 weeks old, purchased from Japan SLC). Seven days later, asolvent group (0-5% CMC, Dai-ichi Kogyo Seiyaku Co., Ltd) and anuntreated group, as well as the test groups given 10 or 100 mg/kg ofCompound 1 orally, were provided. One hour after administration ofCompound 1, 5 μg/body of lipopolysaccharide (LPS, Sigma Co., Ltd.) wasadministered intravenously to induce acute hepatic failure.

The mice in the respective groups were observed for survival rate till 7days after LPS administration.

The results are shown in FIG. 11.

As the figure shows, the survival rate was 30% for the Compound 1 10mg/kg group (test group) and 70% for the Compound 1 100 mg/kg group(test group). In contrast, the survival rates for the untreated andsolvent groups were 0 and 10%, respectively. These results indicate thatCompound 1 exerts a dose-dependent and excellent life-prolonging effectin the model of acute hepatitis.

Pharmacological Test Example 13 Effect of B16 melanoma cells on anexperimental model of pulmonary metastasis

B16 mouse melanoma cells [Koren, S. and Fleischmann, W. R., J.Interferon Research, 6, 473-482 (198)], 2×10 cells, were suspended inEagle's MEM medium (Nissui Pharmaceutical Co., Ltd.) supplemented with10% FBS (Gibco) to which 3 or 10 μg/ml of Compound 1 was previouslyadded or not added. The suspension was cultured in a 75-cm² flask(Corning) in an incubator (National Appliance Model 15300) at 37° C.under 5% carbon dioxide for 4 days. Then, the cells were washed withDulbecco's PBS solution (Nissui pharmaceutical Co., Ltd.), and 0.05%trypsin (Flow Laboratories) was added to detach the cells from theflask. The cells were washed centrifugally (Hitachi 05PR-22, 1200 rpm, 5minutes) twice and after addition of 0.2% trypan blue (Wako PureChemical Industries Ltd.), viable cells were counted using ahemocytometer (No. J7796, Kayagaki Works) and the cells were dilutedwith Hank's solution (Flow Laboratories) to a concentration of 5×10⁶cells/ml.

The cells obtained as above were transplanted, in a population of 3×10⁵cells each, into groups of 7 C57BL/6 mice (7 weeks old, female,purchased from Charles River Japan, Inc.) through the tail vein. Twelvedays later, the mice were killed by cervical dislocation and the numberof colonies metastatized to the lungs was determined.

The results are shown in FIG. 12 and Table 12 below.

                  TABLE 12    ______________________________________    Group       No. of colonies in lungs                                Mean    SE    ______________________________________    Control group                62, 165, 164, 32, 36, 61                                86.7    25.1    Compound 1  14, 7, 11, 6, 8, 24, 0                                 10.0*  2.9    group    (3 μg/ml)    Compound 1  17, 43, 65, 7, 46, 22, 43                                34.7    7.6    group    (10 μg/ml)    ______________________________________     *: P < 0.05 (Student's ttest)

As seen from the figure and the table, an evident inhibition ofpulmonary metastasis was found in both of the groups in which the cellsincubated in the medium containing 3 or 10 μg/ml of Compound 1 weretransplanted.

The apoptosis regulating composition of the invention can be effectivelyused as a cancer chemotherapeutic agent by virtue of its characteristicapoptosis regulating and cell differentiation inducing activities. Thecomposition is also effective as a therapeutic agent for AIDS, ARC, ATL,other related diseases and HTLV-I-associated diseases by virtue of itsantiretrovirus activity, as a therapeutic agent for autoimmune diseasesby virtue of its life-span prolonging activity in autoimmune diseases,as a therapeutic agent for thrombocytopenia by virtue of its effect inincreasing the expression of platelet associated antigents, as atherapeutic agent for Alzheimer's diseases, as a therapeutic agent forhepatitis by virtue of its life-span prolonging activity in hepatorgy,and as an inhibiting agent of pulmonary metastasis.

What is claimed is:
 1. A method for treatment of autoimmune diseasewhich comprises administering to a host afflicted with autoimmunedisease an anti-autoimmune disease effective amount of at least onecarbostyril derivative represented by general formula (1) or a saltthereof ##STR3## wherein R is a benzoyl group which may optionally besubstituted with a lower alkoxy group on the phenyl ring and thecarbon-carbon bond in the 3 and 4 positions of the carbostyril skeletonis a single or a double bond.
 2. The method according to claim 1,wherein said autoimmune disease is one resulting from apoptosisabnormality.
 3. The method according to claim 1, wherein said autoimmunedisease is rheumatoid arthritis.